Metformin is a Novel Suppressor for Vimentin in Human Gastric Cancer Cell Line

Vimentin, an intermediate filament of mesenchymal cells, is upregulated in epithelial-mesenchymal transition (EMT) and has a main role in cancer metastasis. As a new strategy to control metastatic outgrowth, EMT markers are generally inhibited using some drugs or specific siRNA. In this study, AGS gastric cancer cells were treated with metformin and vimentin-specific siRNA (vim-siRNA) for 48 h. The impact of metformin and vim-siRNA on vimentin downregulation in AGS cells were analyzed by quantitative PCR and Western blot. Following treatment with metformin and vim-siRNA, cell motility, migration and invasion abilities of AGS cells were also analyzed. The results showed that inhibition of vimentin due to metformin was comparable with the vim-siRNA. Furthermore, wound-healing and invasion assays showed a significant decrease in migration and invasion of AGS cells following metformin and vim-siRNA treatment. Our finding for the first time indicated that metformin can be an alternative to specific siRNA for inhibition of vimentin expression and migration of AGS cell line. Taken together, our data indicates that the use of metformin might have a priority to siRNA for inhibition of gastric cancer cell behaviors siRNA is more unstable and expensive than metformin, and needs special vehicles and delivery strategies for efficient transfection of cells. Further in vivo studies can reveal metformin's potential in inhibition of EMT and metastasis of cancer cells.

G VIMENTIN is a key indicator of EMT which facilitates cell motility and invasion (2). VIMENTIN induction in advanced stages of GC is related to diffuse type of disease, lymphatic invasion, lymph node metastasis and poor prognosis of patients (2,3). Recently, VIMENTIN has been introduced as an attractive molecular target for diagnostic and therapeutic approaches.
Hence, further understanding of drugs leading to reduction of this protein in GC cells can provide new therapeutic approaches to deal with this disease. EMT inhibition and reversion is a crucial step in controlling metastatic outgrowth (4,5).
Metformin is a well-known anti-diabetic drug that reduces the incidence of malignancies in patients with diabetes (6). Many studies showed that metformin significantly reduces the occurrence of lung, liver, colon and gastric cancers and related mortality (6)(7)(8). In addition to its anti-tumorigenic effects, recent reports on inhibition of EMT genes by metformin support its potential role in fighting cancer metastases. We previously found a significant decrease in cell migration and invasion of GC cell following metformin treatment (9).
Since the discovery of RNAi, siRNA-based therapies have been introduced into clinical trials for treatment of diseases such as cancer (10 (12). As the best of our knowledge, this is the first report comparing the outcome of two approaches in suppression of invasion features of GC cells.

Cell culture
The AGS cell line was prepared from the National Cell Bank of Iran (Pasture Institute, Tehran, Iran) and cultured in DMEM/F12 medium containing 10% FBS (ThermoFisher Scientific, USA) and 1% penicillin-streptomycin at 37 °C in a CO 2 incubator.

MTT test
The AGS cells were seeded into 96-well plates at a density of 5000 cells/well. The MTT assay was done as we previously published (9).

Cell migration assay
The cells were seeded in 6-well plates and cultured till grow as confluent monolayer. for two days.

Cell invasion assay
The AGS cells were starved in a serum-free medium one day prior to beginning the assay.
Culturex 96 well BME invasion assay kit (R&D systems, USA) was used for invasion assay.
Briefly, top chamber was coated with 0.5 X BME  (14). The sequences of primers were presented in Table 1.

Western blotting
Total protein was extracted from controls and metformin/siRNA treated cells by RIPA

Statistical analysis
All experiments were repeated three times and the statistical analyses were performed using ANOVA or Student's t-test. A P-value <0.05 was considered significant.   (Figure 1c).

Metformin and VIM-siRNA inhibited cell migration and invasion
Wound healing progress in control culture was rapidly disappeared following 48 h. However, the healing process was inhibited and the closure of denuded area progressed more slowly in metformin and siRNA treatment groups (P≤0.0001) (Figure 2a and b).
The AGS cells plated into transwell chamber, and the number of cell migrated was measured after 24 h. As shown in Figure 3, metformin and siRNA treatment significantly decreased the invasion ability of AGS cells (P≤0.0001).

Fig. 2. Effect of metformin on the migration of AGS cells compared with siRNA-VIM treated. (a) AGS cells were treated with metformin and siRNA for 48 hours (Magnification 40X). (b)
After 48 hours in metformin treated 76% and in siRNA treated 56% of scratch was filled. Cell migration was quantified by wound-healing assay using Image J software. ***P≤0.0001.

Fig. 3. Effect of metformin on the invasion of AGS cells compared with siRNA-VIM treated. AGS cells were treated with metformin
for 24 hours and the cell invasion was measured in the Culturex 96 well BME invasion assay kit by counting the number of cells invading underside of BME. ***P≤0.0001.  (21). Therefore, at the third day, transcript of VIMENTIN was not detected by qPCR, while a part of earlier expressed proteins was detected by western blot.
In conclusion, our finding for the first time